Compositions and methods for enhancing contrast in imaging
Example compositions of liposomes with hydrophilic polymers on their surface, and containing relatively high concentrations of contrast-enhancing agents for computed tomography are provided. Example pharmaceutical compositions of such liposomes, when administered to a subject, provide for increased contrast of extended duration, as measured by computed tomography, in the bloodstream and other tissues of the subject. Also provided are example methods for making the liposomes containing high concentrations of contrast-enhancing agents, and example methods for using the compositions.
Some medical X-ray imaging techniques provide for detecting variations in contrast of regions of interest of a subject, including different organs, tissues, cells and the like. In order to increase the contrast of regions of interest, some of the imaging techniques may provide for administration of contrast-enhancing agents to the subject. The contrast-enhancing agents may accentuate existing differences in contrast between different areas of interest, or may produce differences in contrast where such differences do not exist without use of the agents.
There have been advancements in medical X-ray imaging, specifically relating to the instruments or machines used to detect the differences in contrast. Such advancements include increases in the speed of such instruments, increases in the resolution of such instruments, and the like. These advancements have provided, in part, for new medical imaging methods. One such method, whole-body imaging, may provide information on the vasculature of the entire body of a subject.
Compared to advances in the instruments used for X-ray imaging, advances in contrast-enhancing agents have not been as forthcoming. Current contrast-enhancing agents for medical imaging using X-rays may have limitations for applications such as whole-body imaging due to, among other things, their rapid clearance from the body, prevalent extravasation, their renal toxicity and their inability to target specific areas of the body.
BRIEF DESCRIPTION OF THE DRAWINGSIn the accompanying drawings, which are incorporated in and constitute a part of the specification, embodiments of contrast-enhancing agent formulations, pharmaceutical compositions containing the formulations, methods for making the formulations and methods for using the formulations in imaging are illustrated which, together with the detailed description given below, serve to describe the example embodiments of formulations, compositions, methods, and so on. It will be appreciated that the embodiments illustrated in the drawings are shown for the purpose of illustration and not for limitation. It will be appreciated that changes, modifications and deviations from the embodiments illustrated in the drawings may be made without departing from the spirit and scope of the invention, as disclosed below.
Definitions
Definitions of selected terms or phrases are contained immediately following, and throughout the disclosure. The definitions include examples of various embodiments and/or forms of components that fall within the scope of a term and that may be used for implementation. The examples are not intended to be limiting and other embodiments may be implemented. Both singular and plural forms of all terms fall within each meaning.
“X-ray imaging,” as used herein, generally refers to any of a number of procedures using a source producing X-rays. Examples of X-ray imaging include computed tomography and the like.
“Computed tomography” or “CT” or “CAT,” as used herein, generally refers to procedures using a rotating X-ray instrument or machine to produce X-ray radiation and direct it through areas of a subject as the instrument rotates. The radiation that is not absorbed by the subject generally is detected and recorded. Generally, the data are sent to a computer which creates detailed cross-sectional images, or slices, of organs and body parts based on differential absorption of X-rays by different areas of the subject.
“Whole body imaging,” as used herein, generally refers to methodologies for obtaining images, using CT for example, of the entire body of a subject. In one type of whole body imaging, the entire vasculature system may be examined. Generally, imaging where the vasculature system is examined is called “blood pool imaging.”
Description
This application describes example compositions comprising liposomes which contain or are associated with one or more contrast-enhancing agents. In one embodiment, the liposomes contain or are associated with relatively high concentrations of contrast-enhancing agents. In one example, the liposomes contain one or more contrast-enhancing agents for X-ray imaging, CT for example. In one example, the contrast-enhancing agents are not radioactive.
In one embodiment, the liposomes have one or more hydrophilic polymers attached to or associated with the liposomes. In one example, the hydrophilic polymers are attached to or associated with the surface of the liposomes. When administered to a subject, the liposomes may provide increased contrast in the body of a subject. In one example, the increased contrast lasts for an extended period of time.
This application also describes example pharmaceutical compositions that contain the liposomes and contrast-enhancing agents, and example methods of making the compositions of liposomes containing contrast-enhancing agents. The application also describes example methods of using the compositions in X-ray imaging.
Contrast-Enhancing Agents
“Contrast-enhancing agent,” as used herein, generally refers to a substance that affects the attenuation, or the loss of intensity or power, of radiation as it passes through and interacts with a medium. It will be appreciated that contrast-enhancing agents may increase or decrease the attenuation. Generally, the contrast-enhancing agents referred to herein increase the attenuation of radiation. In one embodiment, the contrast-enhancing agents described herein are contrast-enhancing agents for X-ray imaging. In one embodiment, the contrast-enhancing agents are used for CT. In one embodiment, the contrast-enhancing agents used herein are nonradioactive. In one embodiment, the contrast-enhancing agents contain iodine.
The contrast-enhancing agents may be classified in various ways. In one classification, for example, iodinated contrast-enhancing agents may be water soluble (e.g., monoiodinated pyridine derivatives, di-iodinated pyridine derivatives, tri-iodinated benzene ring compounds, and the like), water-insoluble (e.g., propyliodone and the like) or oily (e.g., iodine in poppy seed oil, ethyl esters of iodinated fatty acids of poppy seed oil containing iodine, and the like).
In one example, a grouping of iodinated contrast-enhancing agents are water soluble. Present water soluble iodinated contrast-enhancing agents are generally derivatives of tri-iodinated benzoic acid. These compounds may have one or more benzene rings. Such compounds may be ionic or nonionic. Example ionic compounds include acetrizoate, diatrizoate, iodamide, ioglicate, iothalamate, ioxithalamate, metrizoate, sodium meglumine ioxaglate and others. Example, nonionic compounds include metrizamide, iohexol, iopamidol, iopentol, iopromide, ioversol, iotrolan, iodixanol and others.
Liposomes
“Liposomes,” as used herein, generally refer to spherical or roughly spherical particles containing an internal cavity. The walls of liposomes generally are comprised of a bilayer of lipids, particularly phospholipids. There are numerous lipids and phospholipids that can be used to make liposomes. For example, commonly used are amphipathic lipids having hydrophobic and polar head group moieties, and which can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids, or are stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
As used herein, example “phospholipids” include but are not limited to phosphatidic acid (PA) and phosphatidylglycerol (PG), phosphatidylcholine (PC), egg phosphatidylcholine (EPC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). The vesicle-forming lipids of this type may be lipids having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and sphingomyelin (SM), and others. These phospholipids may be fully saturated or partially saturated. They may be naturally occurring or synthetic. In another example, other lipids that can be included in the liposomes are glycolipids.
The phospholipids used in the example liposomes described herein may be those where the two hydrocarbon chains are between about 14-24 carbon atoms in length, and have varying degrees of unsaturation. Some examples of such phospholipids are given below, in Table 1. Although the phospholipids listed below may be used, alone or in combination with other phospholipids, the list is not intended to be complete. Other phospholipids not listed may also be used.
The liposome composition may be formulated to include amounts of fatty alcohols, fatty acids, and/or cholesterol esters or other pharmaceutically acceptable excipients. For example, the liposomes may include lipids that can stabilize a vesicle or liposome composed predominantly of phospholipids. For example, cholesterol between about 25 to 40 mole percent may be used.
In one embodiment, the type of liposomes used are “sterically stabilized liposomes.” Sterically stabilized liposomes generally include a surface that contains or is coated with flexible water soluble (hydrophilic) polymer chains. Such polymer chains may prevent interaction between the liposomes and blood plasma components, the plasma components playing a role in uptake of liposomes by cells of the blood and removal of the liposomes from the blood. Sterically stabilized liposomes may avoid uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen (reticulendothelial system or RES). Such sterically stabilized liposomes may also be called “long circulating liposomes.”
Sterically stabilized liposomes may contain lipids or phospholipids which are derivatized with a polymer chain. The lipids or phospholipids which may be used generally are any of those described above. One exemplary phospholipid is phosphatidylethanolamine (PE) with a reactive amino group which is convenient for coupling to the activated polymers. An exemplary PE is distearyl PE (DSPE).
Examples of polymers which may be suitable for use in sterically stabilized liposomes include the hydrophilic polymers polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose. Polylysine may be used. Lipid-polymer conjugates containing these polymers attached to a suitable lipid, such as PE, may be used. Other example polymers may be used.
In one embodiment, the polymer in the derivatized lipid or phospholipid is polyethylene glycol (PEG). The PEG may have any of a variety of molecular weights. In one example, the PEG chain has a molecular weight between about 1,000-10,000 daltons. Once a liposome is formed, the PEG chains generally provide a surface coating of hydrophilic chains sufficient to extend the blood circulation time of the liposomes in the absence of such a coating. Such liposomes may be called “PEGylated liposomes.” PEGylated liposomes include so-called STEALTH® liposomes, provided by ALZA Corporation.
PEGylated liposomes may also include liposomes with PEG on their surface, where the PEG may be released from the liposome at some time after administration of the liposomes to a subject. In one embodiment, there may be one or more bonds or linkages attaching the PEG, or other hydrophilic polymer, to the liposome surface and/or lipid molecules comprising the liposome surface. In one embodiment, the bonds or linkages may be cleaved, providing for separation of the PEG from the liposome. For example, PEG may be attached to a lipid by one or more disulfide bonds. The disulfide bonds may be cleaved by free thiol, releasing the PEG from the liposome. Other types of cleavable links or bonds may be used to attach the polymers to the liposomes. Other types of agents or compounds may be used to cleave the bonds or linkages.
In one example, the liposomes used have a composition of between about 60 and 75 mole % of one or more of the phospholipids with carbon chains between about 14-24 in length, as described above. A fraction of these phospholipids may be attached to one or more hydrophilic polymers such that between about 1 and 20 mole % of the liposome composition is phospholipid derivatized with polymer chains. In addition, the liposomes used may have between about 25 and 40 mole % cholesterol, or fatty alcohols, fatty acids, and/or other cholesterol esters or other pharmaceutically acceptable excipients, generally for the purpose of stabilizing the liposomes.
In another embodiment, the liposomes may have a molecule or molecules, commonly called a “ligand,” accessible from the surface of the liposome, that specifically binds or attaches to, for example, one or more molecules or antigens. Such ligands may direct or target the liposomes to a specific cell or tissue and may bind to a molecule or antigen on or associated with the cell or tissue. The ligand may be an antibody or antibody fragment. The antibody may be a monoclonal antibody or fragment. Such liposomes may be of a type called “targeted liposomes.”
In one example, targeted liposomes have lipids or phospholipids which have been modified for coupling antibody molecules to the liposome outer surface. These modified lipids may be of different types. The modified lipid may contain a spacer chain attached to the lipid. The spacer chain may be a hydrophilic polymer. The hydrophilic polymer may typically be end-functionalized for coupling antibody to its functionalized end. The functionalized end group may be a maleimide group, for selective coupling to antibody sulfhydryl groups. Other functionalized end groups may include bromoacetamide and disulfide groups for reaction with antibody sulfhydryl groups, activated ester and aldehyde groups for reaction with antibody amine groups. Hydrazide groups are reactive toward aldehydes, which may be generated on numerous biologically relevant compounds. Hydrazides may also be acylated by active esters or carbodiimide-activated carboxyl groups. Acyl azide groups reactive as acylating species may be easily obtained from hydrazides and permit the attachment of amino containing ligands.
In another embodiment, the phospholipid may be modified by a biotin molecule. To attach the antibody molecule to the biotinylated liposome surface, once the liposome is formed, the antibody molecule may also be modified with biotin and then incubated in the presence of the avidin. Biotinylated lipids, such as biotinylated PE, may be commercially available.
In another example, lipids may be modified by a substrate for use in binding a targeting molecule to a liposome surface. Typically, substrates, as exemplified with biotin, are relatively small, less than about 5,000 daltons for example, to allow their incorporation into multilamellar liposomes with a minimum of disruption of the lipid bilayer structures. The substrate may be one capable of binding irreversibly to a targeting molecule, to ensure that the targeting molecule remains bound to the liposomes over its lifetime in the bloodstream.
Preparation of Liposomes Containing Contrast-Enhancing Agents
The liposomes generally may be prepared by a variety of methods. Example methods include, but are not limited to, hydration of dried lipids, introduction of a volatile organic solution of lipids into an aqueous solution causing evaporation of the organic solution, and dialysis of an aqueous solution of lipids and detergents or surfactants to remove the detergents or surfactants. Additional methods are described in U.S. Pat. No. 5,049,389 by Radhakrishnan, entitled “Novel Liposome Composition for the Treatment of Interstitial Lung Diseases,” the descriptions of which are incorporated herein in their entirety by reference.
Liposomes may contain or are associated with one or more contrast-enhancing agents. In one embodiment, the liposomes contain the contrast-enhancing agents. In the process of making liposomes, the contrast-enhancing agents can be added at any desired time. For example, contrast-enhancing agents may be associated with components of liposomes before liposomes are formed. Contrast-enhancing agents may be combined with liposome components at the time the liposomes are made. Contrast-enhancing agents may also be added after the liposomes are formed. Other methods of associating contrast-enhancing agents with liposomes may exist. Generally, contrast-enhancing agents which are hydrophilic in nature are located or associated with the internal cavity of the liposome particles. Contrast-enhancing agents which are lipophilic in nature generally are located or associated with the lipid bilayer of liposome particles. Generally, the contrast-enhancing agents herein are located or associated with the internal cavity of the liposome. The example liposomes may contain at least 30 mg iodine/milliliter (I/ml) of liposome suspension when iodinated contrast enhancing agents are used.
There are a variety of methods known in the art for loading the contrast-enhancing agents into the liposomes. For example, in passive loading, liposomes are formed in a solution of the contrast-enhancing agent to be used. Illustrated in
In active or remote loading, a variety of methods can be used. Generally, in these methods, the agent to be encapsulated by the liposomes (i.e., contrast-enhancing agents) is drawn or attracted into liposomes, or trapped or contained in liposomes as they are formed. In one example,
In another type of active loading, called pH based loading, a proton may be dissociated from the contrast-enhancing agents, causing the contrast-enhancing agents to enter into and remain in the liposomes (see Ceh and Lasic, 1997, J. Colloid Interface Sci., 185:9-18). In still another method, a chelating agent within the liposome may result in trapping of contrast-enhancing agents therein as well as further diffusion of contrast-enhancing agents into the liposomes (see Patent No. WO0023052 by Colbern, Working and Slater, entitled “Liposome-Entrapped Topoisomerase Inhibitors”).
After the loading of contrast-enhancing agents into liposomes, steps may be used to remove contrast-enhancing agents that have not been loaded and are not associated with liposomes, or other impurities. Such steps may comprise techniques such as ion exchange, diafiltration, washing of the liposomes using ultracentrifugation, dialysis, and the like.
After liposomes are made, techniques for manipulating the liposomes may be used. For example, a preparation of liposomes made by standard techniques may vary in size and lamellarity (i.e., wall thickness) after it is made. Techniques such as subjecting the liposomes to a high shearing force, extrusion of the liposomes through membranes, or sonication of the liposomes may be used either to select liposomes of a desired size or modify the liposomes such that they have a desired size. After manipulation of liposomes by these methods, the size distribution of the liposomes may be measured to ensure that liposomes of the desired size have been obtained. Techniques such as Fraunhofer diffraction and dynamic light scattering (DLS) may be used to measure the size distribution of the liposomes. Such techniques generally measure an equivalent spherical diameter which, in the case of Fraunhofer diffraction, may be the diameter of a sphere with the same light scattering properties as the measured liposomes. In the case of DLS, equivalent spherical diameter may be the diameter of a sphere with the same diffusion coefficient as the measured liposomes. Generally, the example liposomes have an average diameter of less than 150 nm. Example preparations of liposomes may have an average diameter of approximately 120 nm or less. Example preparations of liposomes may have an average diameter of approximately 100 nm or less. It will be appreciated that other sizes can be used.
In one embodiment, a nano-scale liposomal formulation carrying over 30 mg of iohexol per ml of liposome can be formulated using passive loading. In this formulation, the lipid composition of the bilayer has been adjusted as described below to allow this amount of contrast-enhancing agent to be encapsulated. In one example, it has been found that by using pure DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) of C16 chain length, with about 40 mole % cholesterol and 5 mole % mPEG-DSPE (N-(carbonylmethoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine) (the polyethylene glycol-conjugated lipid that confers long circulating properties), the encapsulation of active molecules inside the liposomes may be increased by 20% over what is possible using hydrogenated Soy PC(HSPC), a mixture of C16 and C18 lipids, or pure DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) of C18 chain length. Using a formulation of 55 mole % DPPC, 40 mole % cholesterol and 5 mole % mPEG-DSPE and an iohexol solution of 350 mg I/ml, an overall concentration of over 30 mg I/ml has been achieved, with an average liposomal diameter of 100.6±3 nm, as determined by DLS.
Pharmaceutical Compositions and Administration to Subjects
The liposomes containing or associated with one or more contrast-enhancing agents may be part of a pharmaceutical composition suitable for administration to a subject. The compositions generally are administered using a route that delivers the composition to an area of interest. In one example, the compositions of contrast-enhancing agents are administered parenterally to the subject, such as through intravenous, intraarterial, subcutaneous or other route of injection.
The formulation of the particular pharmaceutical composition generally will depend on the method by which the composition is administered to a patient. It will be appreciated that the pharmaceutical compositions can include salt, buffering agents, preservatives, other vehicles and, optionally, other agents. Compositions suitable for parenteral administration may comprise a sterile, pyrogen-free, aqueous or oleaginous preparation which is generally isotonic with the blood of the subject. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Among acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride or other salt, dextrose, phosphate buffered saline and the like, or combinations thereof.
The pharmaceutical compositions used may also contain stabilizers, preservatives, buffers, antioxidants, or other additives. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulations suitable for the administrations may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The pharmaceutical compositions may conveniently be presented in unit dosage form.
Parenteral administration contemplates the use of a syringe, catheter or similar device, which delivers the pharmaceutical composition to a site. Delivery may result, at least initially, in the pharmaceutical composition being systemically distributed throughout the circulatory system of the subject.
Generally, the pharmaceutical compositions may be administered to the subject at a point in time before the imaging of the subject is performed. The amount of the pharmaceutical compositions administered preferably results in increased contrast of one or more tissues of the subject. Ultimately, the attending physician or technician generally will decide the amount of pharmaceutical composition to administer to the subject. Generally, the increase in contrast can be any level above what is present without use of the contrast-enhancing agents in the pharmaceutical compositions. Example increases in contrast of at least 50 HU, at least 100 HU or more, to one or more organ systems, including the vasculature, may be obtained.
Applications
The compositions of liposomes containing contrast-enhancing agents or pharmaceutical compositions thereof, when administered to a subject, may maintain a level of contrast-enhancing agent in the blood and/or organs of a subject that results in an increased contrast and is detectable by X-ray imaging techniques. The increase in contrast may be detectable for an extended period of time. Depending on the particular application, the compositions described herein may have half lives in the circulation of from minutes to hours, to even days. In one example, half lives in the circulation of from 8 to 24 hours can be obtained. In one example, an administered composition provides an enhanced contrast that remains detectable at least 30 minutes after administration. In another example, an administered composition provides an enhanced contrast that remains detectable at least 5 minutes after administration. Many applications, including those in anatomic, functional and molecular imaging may be possible. For example, use of the compositions described herein may have applications in cardiology, oncology, neurology and other areas.
In one embodiment, blood pool imaging may be used to detect and, in some cases, quantify ischemia. For example, because injection of the pharmaceutical compositions generally alters the contrast of the entire vasculature, reduced blood flow as is present in ischemia may be detected. A variety of types of ischemia may be detected, including that causing ischemic bowel disease, pulmonary embolism, and types of ischemia that produce cardiomyopathy, and others. In other applications, aneurysms may also be detected.
In one embodiment, the compositions described herein may be used in cardiac imaging to detect, examine and/or assess stenosis, and the therapy or remediation of stenosis, as occurs in angioplasty, for example. The utility of such techniques may be enhanced through the use of contrast-enhancing agent preparations, such as those described herein.
In one embodiment, the compositions described herein may be used to detect myocardial microcirculatory insufficiencies. Myocardial microcirculation is known to display signs of obstruction before the epicardial arteries show signs of obstruction. Therefore, detection of obstruction in the myocardial microcirculation may be an earlier detector of atherosclerosis in presymptomatic, at-risk patients, than conventional methods. The compositions described herein may facilitate detection of obstructions in the myocardial microcirculation.
In another embodiment, the compositions described herein may be used to detect and characterize a wide range of tumors and cancers. These applications may be facilitated by the property of sterically stabilized liposomes being present for extended periods of time in the circulation and to extravasate at regions where the vasculature is “leaky,” such as in tumors, for example. The leakiness of the vasculature in tumors may be attributed to the high proportion of neovasculature, the result of continuing angiogenesis as the tumor grows in size. Upon encountering such leaky vasculature, liposomes may leave the circulation, driven with the extravasate fluid, by hydrostatic pressure. Such liposomes generally do not return to the circulation after extravasation since the pressure gradient opposes such return. Such methods can be used to detect both primary and metastatic tumors.
In other embodiments, the compositions may be used for “staging” or classification of tumors. Such applications may depend on, among other things, differences in the “leakiness” of the vasculature of a given tumor or cancer at different stages of progression.
In one embodiment, the compositions may be used in the area of monitoring and characterizing injury and healing of damaged spinal cords. In a typical spinal cord injury, as occurs in an automobile accident for example, there may also be damage to tissue surrounding the spinal cord. It is thought that the process of healing of the surrounding tissue may be deleterious to healing of the spinal cord. It is thought that formation of neovasculature in the surrounding tissue, as occurs in healing of the surrounding tissue, may inhibit healing of the spinal cord. It is thought that by inhibiting healing of the surrounding tissue, and the formation of neovasculature in the surrounding tissue, the spinal cord may heal. Subsequently, the surrounding tissue may heal. The compositions of contrast-enhancing agents described here may be useful for monitoring the healing and inhibition of healing of the tissue surrounding the spinal cord.
There may be a variety of other applications for the compositions described herein. For example, the compositions may be used in detection and monitoring of inflammation, reperfusion injuries, and the like.
Additionally, the liposomes which comprise the compositions of contrast-enhancing agents may be targeted to desired cells and tissues in the body of a subject by, for example, attaching antibodies to the surface of the liposomes. Such targeting may result in enhanced contrast to the targeted areas of the body.
The compositions of contrast-enhancing agents may have a relatively long residence time in the body, low extravasation, except in those areas of the vasculature that are leaky as described above, may be relatively nontoxic to the kidneys and may be used to target specific areas of the body. Additionally, the traditional osmolality related toxicity problems associated with ionic contrast-enhancing media generally are not an issue with the liposomal encapsulates since the high osmolality phase is interior to the liposomes and generally is not exposed to the blood.
EXAMPLES Example 1 Preparation of PEGylated Liposomes Containing IohexolExample liposomal iohexol formulations can be produced as follows. Briefly, a lipid mixture (200 mM) of 1,2-Dipalmitoly-sn-glycero-3-phosphocholine (DPPC), cholesterol (chol) and N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-MPEG2000), in a 55:40:5 molar ratio, was dissolved in ethanol at 65° C. The ethanol solution was then hydrated with iohexol (350 mg I/ml) for 1.5-2 hours. Liposomes were extruded on a 10 ml Lipex Thermoline extruder (Northern Lipids, Vancouver, British Columbia, Canada) with 5 passes through a 0.2 μm Nucleopore membrane (Waterman Inc., Newton Mass.) and 7 passes through a 0.1 μm Nucleopore membrane (Waterman Inc., Newton Mass.). Liposomes were then be dialyzed in a 300,000 molecular weight cutoff (MWCO) dialysis bag against phosphate buffer saline (PBS) overnight to remove the free iohexol.
The size of the resulting example liposomal iohexol formulations can be determined by dynamic light scattering (DLS) using a modified BI-90 goniometer, a JDS uniphase 532 nm laser, Hamamastu photomultiplier and Brookhaven DLS Software Version 3.16. The average diameter of the liposomal iohexol capsules was 100.6 nm (STD=3.0 nm), which is in nano-scale range, as determined by DLS.
The iohexol concentrations of example liposomal iohexol formulations can be determined by measuring the absorption at 245 nm using a UV-Vis spectrophotometer. Equivalent iodine concentrations can then be calculated. In the example preparations, different lipid hydration times (1.5 hours and 2 hours) resulted in different iohexol loading concentrations (30 and 34.8 mg I/ml respectively). The 30 mg I/ml iohexol liposomal formulation was used in the in vitro stability tests described below, and the 34.8 mg I/ml iohexol liposomal formulation were used in the in vivo CT imaging experiment described below.
The osmolarity of liposomal iohexol formulation can be measured by, for example, Vapro® vapor pressure osmometer (Wescor Inc.). The osmolarity of the example iohexol formulations ranged between 305 to 315 mmol/kg.
Example 2 In Vitro Stability of PEGylated Liposomes Containing IohexolThe in vitro stability of example liposomal iohexol formulations can be determined by measuring the leakage of iohexol from liposomal iohexol formulations both in PBS at 4° C. and in plasma at 37° C. In the procedure, 1 ml of an example liposomal iohexol formulation was placed in a 300,000 MWCO dialysis bag and dialyzed against 250 ml PBS at 4° C. At each time point (0, 1, 2, 3, 8, 24 hours, and 3, 4, 5, 6, 8, 10, 18 days), 1 ml of the dialysate was removed for a UV absorption-based iohexol measurement. At least three data points were obtained at each time point. After measurement, samples were returned to the PBS to maintain constant volume.
To measure stability in plasma, the example liposomal iohexol formulations can be dialyzed against 250 ml PBS at 25° C. for 1 hour to remove the free iohexol. In these experiments, 1 ml liposomal iohexol formulations was placed in a 300,000 MWCO dialysis bag with 4 ml of human plasma, and dialyzed against 250 ml PBS at 37° C. (1:4 ratio was chosen). One ml of the external phase was removed at 0, 1, 2, 3, 4, 5, 6 and 8 hours respectively, and analyzed by the UV-vis absorption. Since plasma components also leak from the dialysis bag and have a finite absorbance at 245 nm, a control experiment, where a PBS-plasma mixture is dialyzed against PBS, was also performed. The absorbance of the external phase was subtracted from that for the liposomal iohexol formulation experiments and the resulting absorbance traces can be representative of the leakage of iohexol from liposomal iohexol formulations. The results showed that the liposomal iohexol formulation was stable in PBS and in human plasma.
The example leakage curves of iohexol 300 is shown in
The leakage curves of an example iohexol-plasma mixture 400 is shown in
A female rabbit weighing 2.2 kg was anesthetized with 35 mg/kg ketamine and 5 mg/kg xylazine given intramuscularly, followed by 2% isoflurane vapor given by face cone. After tracheal intubation and placement of venous catheter in an ear vein, 20 mg pentobarbital was given intravenously. The animal's lungs were ventilated using a pressure control ventilator set to peak airway pressure of 15 cm H2O, and 25 breaths min−1. After transport to the CT scanner, the animal was given 0.25 mg of pancuronium (muscle relaxant) to insure minimal motion during the image acquisition. Supplemental pentobarbital was given every 30-60 minutes, 10-20 mg per dose. An initial volume image of the chest and abdomen was obtained using a 4 slice Phillips MX8000 MDCT scanner in spiral scanning mode, (100 mAs, 120 keV) with a single slice equivalent pitch of 1.25, and a slice collimation and thickness of 1.3 mm. Images were reconstructed into a 512×512 matrix using a standard reconstruction kernel (the “B” kernel). A 0.5 second gantry rotation speed was used. During each imaging protocol, the rabbit was held apneic with airway pressure fixed at 20 cm H2O (e.g. near total lung capacity) using an underwater bubbler tube on the exhalation port. Next, 15 ml of 34 mg I/ml liposomal iohexol formulation was hand-injected followed by a repeat volume image, then a second injection of 15 ml of liposomal iohexol formulation suspension was followed by a third volume image. A total dose of 475 mg iodine per kg was given in the two injections. Repeat volume images were then initiated at approximately 12, 60, 90, 120, 150 and 180 minutes after the second contrast injection. Following the last image acquisition (˜3.5 hr post injection of contrast agent), the animal was euthanized with an overdose of pentobarbital and a final, high resolution image was obtained with no motion artifact (with the same airway pressure and image acquisition settings). Finally, an ultrahigh resolution scan was obtained using an ultra sharp reconstruction kernal (“D” kernal and a 1024×1024 image matrix) to evaluate anatomic detail without the presence of cardiogenic motion.
Example 4 Image ReconstitutionSubsequent offline example reconstructions were performed for each of the scans obtained as described in Example 3 with the smallest field of view (5 cm×5 cm, 0.1 mm voxel size) for 3D viewing of the heart. The enhanced heart chambers were visualized by selecting appropriate settings of the volume rendering software present on the Philips MXV workstation software (version 4.1). Once the settings were established, the same rendering and display settings were used for all time points. Additional structures were segmented at various time points.
Quantitative analysis was performed by locating regions of interest (ROI) in the aorta, heart, kidney (core and cortex), liver, muscle and spleen. Mean Hounsfield units (HU) were determined at each time point to enable tracking of any decay in contrast concentration with time in each of these structures. Slice and slice location of the ROI's were adjusted for minor variations in anatomic configuration of the rabbit from time point to time point.
Example 5 Time-Attenuation of PEGylated Liposomes Containing Iohexol In Vivo The example image analysis described in Example 4 was performed at regions of interest in the aorta, kidney (medulla and cortex), liver parenchyma, back muscle, left main coronary artery, pulmonary artery, and in the main stem bronchus (as a control value) and plotted over time in a graph 500 (
These data indicate the residence time of example PEGylated liposome formulations, which provided contrast enhancement, to be more than 3 hours. Additionally, the data show that contrast enhancement in muscle can be low, indicating the liposomal iohexol can be retained in the blood vessels and does not rapidly extravasate. Additionally, the contrast enhancement in the liver parenchyma indicated that clearance of the composition may substantially be due to the liver, and not the kidneys.
Example 6 In Vivo Images of Heart after Administration of PEGylated Liposomes Containing Iohexol Additionally, example images of the rabbit heart were analyzed 700 (
The above descriptions have referred to the preferred embodiments and selected alternate embodiments. Modifications and alterations will become apparent to persons skilled in the art upon reading and understanding the preceding detailed description. It is intended that the embodiments described herein be construed as including all such alterations and modifications insofar as they come within the scope of the appended claims or the equivalence thereof.
Claims
1. A composition for enhancing contrast of one or more areas of a subject for X-ray imaging when administered to a subject, comprising:
- sterically stabilized liposomes containing or associated with one or more nonradioactive contrast-enhancing agents.
2. The composition of claim 1, where the X-ray imaging is computed tomography.
3. The composition of claim 1, where the contrast-enhancing agents are iodinated ionic or iodinated nonionic compounds.
4. The composition of claim 3, where a suspension of the sterically stabilized liposomes has a concentration of at least 30 milligrams of iodine per milliliter of the suspension.
5. The composition of claim 1, where an average diameter of the liposomes in the composition is less than about 150 nanometers.
6. The composition of claim 1, where an average diameter of the liposomes in the composition is less than about 120 nanometers.
7. The composition of claim 1, where the composition is capable of being administered to the bloodstream of the subject.
8. The composition of claim 7, where the composition provides an enhanced contrast that remains detectable at least 30 minutes after administration.
9. The composition of claim 7, where the composition provides an enhanced contrast in at least part of a vasculature or an organ of a subject that is increased by at least 50 Hounsfield units.
10. The composition of claim 1, where the sterically stabilized liposomes are PEGylated liposomes.
11. The composition of claim 1, where the sterically stabilized liposomes are targeted liposomes.
12. A composition for use in computed tomography of a subject, comprising:
- one or more iodinated nonradioactive contrast-enhancing agents contained within or associated with PEGylated liposomes, where intravenous administration of an amount of the composition to the subject provides enhanced contrast of at least 50 Hounsfield units in at least part of a vasculature or an organ of the subject at least 30 minutes after administration.
13. The composition of claim 12, where a milliliter of the composition contains at least 30 milligrams of iodine.
14. A pharmaceutical composition, comprising one or more compositions of claim 1, wherein the pharmaceutical composition is in a form that is capable of being injected intravenously into a subject.
15. A method comprising:
- selecting one or more nonradioactive contrast-enhancing agents; and
- forming sterically stabilized liposomes in the presence of the nonradioactive contrast-enhancing agents to provide liposomes containing or associated with one or more contrast-enhancing agents.
16. The method of claim 15, where one milliliter of a suspension of the sterically stabilized liposomes has at least 30 milligrams of iodine.
17. The method of claim 15, where an average diameter of the sterically stabilized liposomes is less than about 120 nanometers.
18. The method of claim 15, where liposomes are formed in the presence of one or more contrast-enhancing agents using a method selected from a group consisting of hydration of dried lipids in the presence of one or more contrast-enhancing agents, mixing a volatile organic solution of lipids with an aqueous solution of one or more contrast-enhancing agents causing evaporation of the organic solution, and dialysis of an aqueous solution of lipids and detergents and/or surfactants to remove the detergents and/or surfactants and form liposomes in the presence of one or more contrast-enhancing agents.
19. A method comprising:
- forming sterically stabilized liposomes; and
- drawing one or more nonradioactive contrast-enhancing agents into the liposomes to provide liposomes containing or associated with one or more contrast-enhancing agents.
20. The method of claim 19, where one milliliter of a suspension of the sterically stabilized liposomes has at least 30 milligrams of iodine.
21. The method of claim 19, where an average diameter of the sterically stabilized liposomes is less than about 120 nanometers.
22. A method of imaging a subject, the method comprising:
- introducing a composition of sterically stabilized liposomes containing or associated with one or more nonradioactive contrast-enhancing agents into the bloodstream of a subject; and
- generating images of a region of interest in the subject where the contrast-enhancing agents cause a contrast enhancement in the region of interest by at least 50 Hounsfield units for a duration of longer than 5 minutes.
23. The method of claim 22, where the generating includes acquiring one or more images by computed tomography.
24. The method of claim 23, where the images are used for one or more of detection, quantification, characterization, classification or monitoring, of ischemia, myocardial microcirculatory insufficiencies, tumors, cancers, healing and inflammation.
Type: Application
Filed: Apr 21, 2004
Publication Date: Oct 27, 2005
Patent Grant number: 7713517
Inventors: Ananth Annapragada (Manvel, TX), Ravi Bellamkonda (Marietta, GA), Eric Hoffman (Iowa City, IA), Chandra Vijayalakshmi (Manvel, TX)
Application Number: 10/830,190
